This invention relates to a method of purifying hyaluronic acid.
Hyaluronic acid (HA) is a biological polymer. It has only two components, D-glucuronic acid and 2-acetamido-2-deoxy-D-glucose, which repeat from 250 to 25,000 times. HA does not maintain a rigid tertiary structure, but instead, typically has a random coil configuration, familiar to most polymer scientists and engineers. The coil may be held semi-rigidly in its configuration by hydrogen bonds, but these are weak and can easily be disrupted. The polymer typically exists at physiological pH, at which the carboxyl groups are dissociated, conferring a polyanionic character to the molecule. In other words, for every disaccharide unit, there is a strong negative charge.
Hyaluronic acid is a critical component of connective tissue and typically exists in a state of strong association with proteins and other glycosoamino glycans (GAGs) such as Heparin and Chondroitin Sulfate. The chemical and physical structure of hyaluronic acid is very well known though the methods by which it interacts with the body are generally less understood.
Despite the fact that the chemical identity of the molecule never changes, (i.e., it always contains the same linear repeating disaccharide units), the number of repeating units can vary greatly by source. The table shows the molecular weight of hyaluronic acid isolated from various sources.
Hyaluronic acid has very unique properties when it is in its solution form. In particular, at very low concentrations in water (xe2x89xa61%) viscosities can exceed 50,000 cps, and the solution has significant non-Newtonian behavior in its elasticity, shear thinning, and cohesiveness. Hyaluronic acid, because it can hold large amounts of water (over 5 times what glycerin holds), has moisturizing and lubricating properties. It is also, when purified properly, biologically inert, which makes it extremely useful for various medical applications.
The major issue with HA production is a lack of technology that removes the lower molecular weight impurities that is fast, economical, and non-destructive to the HA molecule. Below is a description of two methods currently used to make medical grade HA.
In Balazs, U.S. Pat. No. 4,141,973, the HA production process is as follows: slice rooster combs into small pieces, and wash with ethanol (4L ethanol to 1 kg comb). Discard ethanol and repeat until ethanol is not cloudy. Add combs to water and chloroform in the following amounts: 2.5 kg combs: 10 liters water: 0.5 liters chloroform. Mix and allow to combs to swell. The combs are then filtered from the broth. NaCl is added to the broth and additional chloroform extractions are performed. Pronase(copyright) digestion is accomplished, and the Pronase(copyright) is then removed via chloroform extraction and centrifugation. The product is then filtered through sterilizing filters, followed by precipitation in ethanol. The HA is then formulated into medical products.
In Swann, Studies on Hyaluronic Acid: The Preparation of and Properties of Rooster Comb Hyaluronic Acid, Biochim, Biophys. Acta, 156, pp 17-30, 1968, the process described is as follows: slice rooster combs into small pieces, and wash with acetone. Discard acetone and repeat until acetone is not cloudy. Add combs to water with a preservative to prevent microbial growth (EDTA). Combs are then filtered from broth, NaCl is added, and the broth is centrifuged and precipitated in 3 volumes of ethanol. The precipitate is dissolved in a Tris buffer, pH=8.0, and Pronase(copyright) digestion performed, NaCl added, and then precipitated in 3 volumes ethanol. Precipitate was then dissolved in water and extracted against chloroform. The aqueous phase was added to a 1% CPC solution and the resulting precipitate allowed to settle overnight. Precipitate was added to 0.3 M NaCl. The solution was centrifuged and precipitated in three volumes of ethanol. It is assumed that at this point, the hyaluronic acid would be dissolved in water and sterilized using filters and precipitated in ethanol (same as Balazs). The product would then be formulated into medical products.
These methods are used to produce the materials shown in the next table, as reported by Uden and Lavoie, Laboratory Evaluation of Commercial Hyaluronate Sodium Products, Journal of Equine Veterinary Science, Vol. 17(3), pp. 123-125, 1997.
In the table, IV (intrinsic viscosity) is a measure of molecular weight. 750,000 daltons of MW is about equal to an IV of 1750 ml/g.
Balazs"" process lacks a CPC step, which helps remove DNA as evidenced by Swann""s lower UV numbers. It also appears than Swann""s material has a lower protein level.
In addition, both methods take a very long time, a lot of ethanol (a flammable and expensive liquid) and multiple precipitation steps, each requiring the manual handling of the product, to purify a small amount of HA. For example, Swann""s method as practiced in the lab has the following time and ethanol quantity parameters:
The inventive process yields hyaluronic acid from rooster combs having qualities that are equal to, or in some cases superior to, hyaluronic acid derived by the industry standard procedures. This is supported by the following facts. First, the molecular weight of the HA made in accordance with the invention as measured by intrinsic viscosity is similar to that of hyaluronic acid derived by the standard method. Second, the inventive HA solution is as free of protein as other commercially used hyaluronic acid. Third, the UV absorbance at 260 nm is superior to that of other commercial hyaluronic acid solutions with comparable intrinsic viscosities. Lastly, the process takes 55% of the time and only 67% of the solvent required to make hyaluronic acid solutions as compared to the standard process.
Molecular weight is the parameter that most affects the physical properties of hyaluronic acid-based medical devices. Molecular weight influences the viscosity vs. concentration profiles of hyaluronic acid solutions, and plays a major role in its other visco-elastic properties. In particular, solutions that require a given viscosity for therapeutic efficacy require lower concentrations of hyaluronic acid if the molecular weight is increased. This has been shown to provide advantages in both ocular and orthopedic applications. A process that is capable of generating medical grade hyaluronic acid having a molecular weight greater than 750,000 daltons thus has significant commercial potential.
When considering a chemical species for any medical application, one of the key parameters is purity. FDA will not approve products for use in the United States if a certain level of purity is not reached. In addition, the process must be robust enough to guarantee the product is consistently pure. In the HA of the invention, the two major measurements of purity, protein concentration and UV absorbance at 260 nm, either meet or exceed current industry standards for approved products in the U.S.
Finally, the inventive process takes less time to produce hyaluronic acid, and less raw materials. Less labor and material translates into a cost savings in production.
Overall, the advantages of the inventive process are that it:
1. Generates hyaluronic acid that is high in molecular weight so as to be efficacious in medical applications,
2. Meets or exceeds all standards for purity and is therefore a strong candidate for FDA approval, and it is
3. Cost effective and competitive.
These qualities clearly demonstrate a superior method of purifying medical grade hyaluronic acid from rooster comb. Those skilled in the art will also recognize the potential for this method to be used for high MW HA from any source, not just rooster combs.
Rooster comb was used in the examples of the invention because it is felt in the industry that rooster comb has more impurities than other HA sources. Thus, the process should be even more effective in purifying HA from other sources.
This invention features a method for purifying high molecular weight hyaluronic acid, comprising the steps of providing one or more raw hyaluronic acid sources; extracting the hyaluronic acid from the sources; precipitating the hyaluronic acid extracts; dissolving extract precipitates in water; extracting enzyme inhibitors from the dissolved precipitates with chloroform, and centrifuging to isolate an aqueous portion; adding one or more protein hydrolyzing enzymes to the aqueous portion in a reactor; adding a solution of CPC and NaCl to the reactor; filtering reactor contents through at least one membrane filter; filtering membrane filtered solution through at least one diafilter having a molecular weight cutoff of about 30 kDa; precipitating diafiltered solution and isolating purified hyaluronic acid precipitate; and formulating isolated precipitate to about 10 mg/ml.
The aqueous portion is preferably maintained in the reactor with the hydrolyzing enzymes at a pH of about 8.0 using a buffer. The aqueous portion may be maintained in the reactor with the hydrolyzing enzymes at a temperature of about 37xc2x0 C. Also claimed is a composition comprising hyaluronic acid purified using the described methods.
The purified hyaluronic acid preferably has an average molecular weight greater than about 750,000 Daltons. The solution of CPC and NaCl preferably comprises about 2% CPC and about 0.3 M NaCl.
Also featured is a method for purifying high molecular weight hyaluronic acid, comprising the steps of disinfecting one or more raw hyaluronic acid sources; placing the disinfected source in water with an antiseptic to form a solution; mixing the solution until the solution forms an extract having a viscosity of at least about 500 cps; straining the extract from the solution and treating with NaCl; centrifuging the treated extract and adding ethanol to form a precipitate; dissolving the precipitate in water; extracting enzyme inhibitors from the dissolved precipitate with chloroform and centrifuging to produce an aqueous portion; adding one or more protein hydrolyzing enzymes to the aqueous portion in a reactor; adding a solution of CPC and NaCl to the reactor; filtering reactor contents through at least one membrane filter; filtering membrane filtered solution through at least one diafilter having a molecular weight cutoff of about 30 kDa; precipitating diafiltered solution and isolate purified hyaluronic acid precipitate; and formulating isolated precipitate to about 10 mg/ml.